Identification and recovery of minor HIV-1 variants using the heteroduplex tracking assay and biotinylated probes.
We describe a method to identify and recover minor human immunodeficiency virus type 1 (HIV-1) sequence variants from a complex population. The original heteroduplex tracking assay (HTA) was modified by incorporating a biotin tag into the probe to allow for direct sequence determination of the query strand. We used this approach to recover sequences from minor HIV-1 variants in the V3 region of the env gene, and to identify minor drug-resistant variants in pro. The biotin-HTA targeting of the V3 region of env allowed us to detect minor V3 variants, of which 45% were classified as CXCR4-using viruses. In addition, the biotin-protease HTA was able to detect mixtures of wild-type sequence and drug-resistance mutations in four subjects that were not detected by bulk sequence analysis. The biotin-HTA is a robust assay that first separates genetic variants then allows direct sequence analysis of major and minor variants.
Schnell
Gretja
G
Department of Microbiology and Immunology, Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill School of Medicine, Chapel Hill, NC 27599-7295, USA.
Ince
William L
WL
Swanstrom
Ronald
R
D019921:Biotinylation; D024882:Drug Resistance, Viral / Q000235:genetics; D014644:Genetic Variation; D017320:HIV Protease Inhibitors / Q000627:therapeutic use; D015497:HIV-1 / Q000235:genetics*; D020180:Heteroduplex Analysis / Q000379:methods*; D006801:Humans; D015345:Oligonucleotide Probes; D017422:Sequence Analysis, DNA / Q000379:methods*; D054299:env Gene Products, Human Immunodeficiency Virus / Q000235:genetics
D017320:HIV Protease Inhibitors; D015345:Oligonucleotide Probes; D054299:env Gene Products, Human Immunodeficiency Virus